The variations on the theme of protein purification have been explored for more than fifty years. The literature on this subject is extensive and a plethora of techniques is available to the practitioner, including ion exchange chromatography, adsorption chromatography, gel electrophoresis, ammonium sulfate precipitations, and gel filtration. Over the years there have been substantial improvements in the technology of conducting many of the foregoing methods, and in particular, it has been possible to automate and speed up the procedures related to column chromatography and development of electrophoresis gels. Despite these technical advances, and despite the large number of proteins which have been subjected to these procedures, the selection of a successful procedure, or more usually combination of procedures, for a particular protein found in a particular milieu has remained unpredictable, unselectable in advance, and subject to considerable experimentation in each particular case.
Human TNF has been purified as a native protein using culture supernatants from induced HL-60 cells as a source by a combination of anion exchange chromatography and reverse phase HPLC, with elution in a linear gradient of acetonitrile (Wang, A. M., et al Science (1985) 228: 149-154). Similar procedures had been previously employed (Matthews, N., Br J Cancer (1981) 44: 418) without resulting in a homogeneous preparation. However, this technique is not optimally efficient even for the native TNF secreted from, for example, HL-60 or other TNF secreting cell lines, and is inappropriate for recombinantly produced TNF, due to inactivation of biological activity at low pH.
The method of the invention substitutes a hydrophobic chromatographic support for the reverse phase support used previously and permits isolation of pure TNF and various TNF muteins using a decreasing salt concentration gradient. The resulting purified TNF is homogeneous with respect to TNF molecular size but, depending on the particular form of recombinant TNF produced, may contain side chain modifications detectable upon isoelectric focusing or other modifications which alter the isoelectric points.